Many aspects of the mechanisms by which Cu is taken up from the blood plasma by different kinds of cells (including the transporters and delivery proteins involved), and just how (after cell entry) Cu finds its way to specific secretions, remain to be defined. We have obtained evidence that lactation profoundly alters the tissue distribution of newly absorbed Cu, diverting most of it from the liver and kidney to the mammary gland, where it rapidly appears in the milk and in milk ceruloplasmin. Cu in milk ceruloplasmin may be more bio-available to the infant than the other forms of Cu in the milk, which have not been well defined. A polarized cell culture model responsive to lactational hormones has been developed for studying milk production by mammary epithelial cells. Mutant rodent models lacking specific proteins that could be involved in the steps required for uptake of Cu by mammary gland and its delivery to developing milk are available. The long term objective is to understand how Cu distribution and transport are regulated under normal conditions and in gestation and lactation. The immediate objectives are to determine how Cu from the exchangeable plasma pool enters mammary (and hepatic) cells, under normal conditions and in lactation, to explain the marked changes in distribution observed in the latter condition (what potential membrane transporters are involved, and how their expression and/or disposition is changed by lactation). The objectives are also to determine how, after cell entry, copper is routed to the milk and milk cemloplasmin; the role(s) of ATOX1, WND and/or MNK in this process and in regulating the copper content of the milk; and to further define the copper components of mammalian milk. Expression of potential transporters will be measured at the mRNA and protein levels, by Real Time PCR and immunoassays, respectively; and localization will be followed with immunofluorescence and confocal microscopy. Alterations in expression will be induced in the cell culture model with lactational hormones, transfection, and antisense oligos. Effects of natural and knockout mutations of specific transporters in whole animal models on copper distribution and transport will be determined. Cu uptake and its emergence in milk and milk ceruloplasmin will be followed with 67Cu/64Cu and immunoprecipitation. Effects on Cu uptake of specific antibodies against membrane transporters will be measured. Cu binding components in milk secretions will be characterized. It is expected that the proposed studies will greatly advance our knowledge of how Cu is distributed to cells from the blood and particularly how mammary epithelial cells put Cu into components of the milk that may have specific effects on the infant.